Development of Porous Hydrogel Scaffolds with Multiple Cues for Liver Tissue Engineering

  • Kirthanashri Srinivasan Vasanthan
  • Anuradha Subramanian
  • Uma Maheswari Krishnan
  • Swaminathan Sethuraman


Development of tissue-engineered construct for hepatic regeneration remains challenging due to the lack of potential milieu to control the trans-differentiation of hepatocytes. In the present study, galactose containing poly(vinyl alcohol) (PVA) and gelatin (8:2 and 9:1) hydrogels impregnated with hepatocyte growth factor (HGF)-loaded nanoparticles (241 ± 56 nm) using freeze/thaw technique was fabricated. The scaffolds with the HGF encapsulation efficiency of 57 ± 2.9% exhibited two-stage release kinetics for 30 days. Hepatogenic potential of the fabricated scaffolds were evaluated by determining the adhesion, viability, and proliferation with functional activity of rat primary hepatocytes for 28 days. Scaffolds of 8:2 ratios of PVA and gelatin maintained spheroidal morphology of hepatocytes and exhibited significantly higher metabolic secretions for 28 days (p < 0.05). Hence, galactose-based PVA-gelatin (8:2) hydrogel scaffold could be a promising alternative substrate to retain the phenotype and functional characteristics of hepatocytes due to the co-existence of bioactive motif and trophic factors.

Lay Summary

The developed 3D scaffold with growth factors encapsulated nanospheres possesses characteristic features for hepatic regeneration and is proposed to be a candidate material for further invivo evaluation


Hydrogel Hepatocyte growth factor Nanospheres Poly(vinyl alcohol) Poly(lactide-co-glycolide) primary hepatocytes 



The authors wish to acknowledge the Indian Council for Medical Research (35/22/2010-BMS), Nano Mission (SR/S5/NM–07/2006 and SR/NM/PG–16/2007), FIST program—Department of Science & Technology, Govt. of India for funding (SR/FST/LSI–327/2007), and FIST pharmacy program (SR/FST/LSI-058/2010) for cell culture facility. Prof. T. R. Rajagopalan R & D Cell of SASTRA University is also acknowledged.


  1. 1.
    Habibullah CM, Syed IH, Qamar A, Taher-Uz Z. Human fetal hepatocyte transplantation in patients with fulminant hepatic failure. Transplantation. 1994;58:951–2.CrossRefGoogle Scholar
  2. 2.
    Strom SC, Fisher RA, Thompson MT, Sanyal AJ, Cole PE, Ham JM, et al. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation. 1997;63:559–69.CrossRefGoogle Scholar
  3. 3.
    Vasanthan KS, Subramanian A, Krishnan UM, Sethuraman S. Role of biomaterials, therapeutic molecules and cells for hepatic tissue engineering. Biotechnol Adv. 2012;30:742–52.CrossRefGoogle Scholar
  4. 4.
    Cho CS, Seo SJ, Park IK, Kim SH, Kim TH, Hoshiba T, et al. Galactose-carrying polymers as extracellular matrices for liver tissue engineering. Biomaterials. 2006;2:576–85.CrossRefGoogle Scholar
  5. 5.
    Allen JW, Bhatia SN. Engineering liver therapies for the future. Tissue Eng. 2002;8:725–37.CrossRefGoogle Scholar
  6. 6.
    Geckil H, Xu F, Zhang X, Moon S, Demirci U. Engineering hydrogels as extracellular matrix mimics. Nanomedicine (Lond). 2010;5:469–84.CrossRefGoogle Scholar
  7. 7.
    Hago EE, Li X. Interpenetrating polymer network hydrogels based on gelatin and PVA by biocompatible approaches: synthesis and characterization. Adv Mat Sci Eng. 2013;2013:1–8.CrossRefGoogle Scholar
  8. 8.
    Chao YC, Ying L, Zhang PC, Zhuo RX, Kang ET, Leong KW, et al. High density of immobilized galactose ligand enhances hepatocyte attachment and function. J Biomed Mater Res A. 2003;67:1093–104.Google Scholar
  9. 9.
    Underhill GH, Chen AA, Albrecht DR, Bhatia SN. Assessment of hepatocellular function within PEG hydrogels. Biomaterials. 2007;28:256–70.CrossRefGoogle Scholar
  10. 10.
    Tsang VL, Chen AA, Cho LM, Jadin KD, Sah RL, DeLong S. Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels. FASEB J. 2007;21:790–01.CrossRefGoogle Scholar
  11. 11.
    Ambury RF, Merry CRL, Ulijn RV. Sugar functionalised PEGA surfaces support metabolically active hepatocytes. J Mater Chem. 2011;21:2901–8.CrossRefGoogle Scholar
  12. 12.
    Breslow JL, Sloan HR, Ferrans VJ, Anderson JL, Levy RI. Characterization of the mouse liver cell line FL83B. Exp Cell Res. 1973;78:441–53.CrossRefGoogle Scholar
  13. 13.
    Yin C, Liao K, Mao HQ, Leong KW, Zhuo RX, Chan V. Adhesion contact dynamics of HepG2 cells on galactose-Immobilized substrates. Biomaterials. 2003;24:837–50.CrossRefGoogle Scholar
  14. 14.
    Zhu XH, Wang CH, Tong YW. In vitro characterization of hepatocyte growth factor release from PHBV/PLGA microsphere scaffold. J Biomed Mater Res A. 2009;89:411–23.CrossRefGoogle Scholar
  15. 15.
    Xue F, Takahara T, Yata Y, Kuwabara Y, Shinno E, Nonome K, et al. Hepatocyte growth factor gene therapy accelerates regeneration in cirrhotic mouse livers after hepatectomy. Gut. 2003;52:694–700.CrossRefGoogle Scholar
  16. 16.
    Ishii T, Sato M, Sudo K, Suzuki M, Nakai H, Hishida T, et al. Hepatocyte growth factor stimulates liver regeneration and elevates blood protein level in normal and partially hepatectomized rats. J Biochem. 1995;117:1105–12.CrossRefGoogle Scholar
  17. 17.
    Matsumoto K, Nakamura T. Hepatocyte growth factor: molecular structure and implications for a central role in liver regeneration. J Gastroenterol Hepatol. 1991;6:509–19.CrossRefGoogle Scholar
  18. 18.
    Zern BJ, Chu H, Wang Y. Control growth factor release using a self-assembled [polycation:heparin] complex. PLoS One. 2010;5:11017–24.CrossRefGoogle Scholar
  19. 19.
    Hou YT, Ijima H, Matsumoto S, Kubo T, Takei T, Sakai S, et al. Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes. J Biosci Bioeng. 2010;110:208–16.CrossRefGoogle Scholar
  20. 20.
    Lee K, Silva EA, Mooney DJ. Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. J R Soc Interface. 2011;8:153–70.CrossRefGoogle Scholar
  21. 21.
    Kuppan P, Sethuraman S, Krishnan UM. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate)-based nanofibrous scaffolds to support functional esophageal epithelial cells towards engineering the esophagus. J Biomed Sci Polym Ed. 2014;25:574–93.CrossRefGoogle Scholar
  22. 22.
    Jaidev LR, Krishnan UM, Sethuraman S. Gemcitabine loaded biodegradable PLGA nanospheres for in vitro pancreatic cancer therapy. Mater Sci Eng C Mater Biol Appl. 2015;47:40–7.Google Scholar
  23. 23.
    Kuppan P, Sethuraman S, Krishnan UM. Interaction of human smooth muscle cells with nanofibrous scaffolds: effect of fiber orientation on cell adhesion, proliferation, and functional gene expression. J Biomed Mater Res A. 2014; doi: 10.1002/jbm.a.35360.
  24. 24.
    Vasanthan KS, Subramanian A, Krishnan UM, Sethuraman S. Influence of 3D porous galactose containing PVA/gelatin hydrogel scaffolds on three-dimensional spheroidal morphology of hepatocytes. J Mater Sci Mater Med. 2015;25:5345–65.Google Scholar
  25. 25.
    Mao S, Shi Y, Li L, Xu J, Schaper A, Kissel T. Effects of process and formulation parameters on characteristics and internal morphology of poly(d,l-lactide-co-glycolide) microspheres formed by the solvent evaporation method. Eur J Pharm Biopharm. 2008;68:214–23.CrossRefGoogle Scholar
  26. 26.
    Farahani TD, Entezami AA, Mobedi H, Abtahi M. Degradation of poly(D,L-lactide-co-glycolide) 50:50 implant in aqueous medium. Iran Polym J. 2005;14:753–63.Google Scholar
  27. 27.
    Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers (Basel). 2011;3:1377–97.CrossRefGoogle Scholar
  28. 28.
    Lamprecht A, Ubrich N, Hombreiro Pérez M, Lehr C, Hoffman M, Maincent P. Influences of process parameters on nanoparticle preparation performed by a double emulsion pressure homogenization technique. Int J Pharm. 2000;196:177–82.CrossRefGoogle Scholar
  29. 29.
    Bishi DK, Mathapati S, Venugopal JRR, Guhathakurta S, Cherian KM, Ramakrishna S, et al. Trans-differentiation of human mesenchymal stem cells generates functional hepatospheres on poly (L-lactic acid)-co-poly(e-caprolactone)/collagen nanofibrous scaffolds. J Mater Chem B. 2013;1:3972–84.CrossRefGoogle Scholar
  30. 30.
    Kim M, Lee JY, Jones CN, Revzin A, Tae G. Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes. Biomaterials. 2010;31:3596–03.CrossRefGoogle Scholar
  31. 31.
    Brophy CM, Luebke-Wheeler JL, Amiot BP, Khan H, Remmel RP, Rinaldo P, et al. Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function. Hepatology. 2009;49:578–86.CrossRefGoogle Scholar
  32. 32.
    Zeng L, An L, Wu X. Modeling drug-carrier interaction in the drug release from nanocarriers. J Drug Deliv. 2011;2011:370308–23.CrossRefGoogle Scholar
  33. 33.
    Dash S, Murthy PN, Nath L, Chowdhury P. Kinetic modeling on drug release from controlled drug delivery systems. Acta Pol Pharm. 2010;67:217–23.Google Scholar
  34. 34.
    Costa P, Sousa Lobo JM. Modeling and comparison of dissolution profiles. Eur J Pharm Sci. 2001;13:123–33.CrossRefGoogle Scholar
  35. 35.
    Manish J, Abhay K. Sustained release matrix type drug delivery system: a review. J Drug Del Ther. 2012;2:142–8.Google Scholar
  36. 36.
    Ramana LN, Sharma S, Sethuraman S, Ranga U, Krishnan UM. Evaluation of chitosan nanoformulations as potent anti-HIV therapeutic systems. Biochim Biophys Acta. 1840;2014:476–84.Google Scholar
  37. 37.
    Sharma R, Walker RB, Pathak K. Evaluation of the kinetics and mechanism of drug release from econazole nitrate nanosponge loaded carbapol hydrogel. Ind J Pharm Edu Res. 2011;45:25–31.Google Scholar
  38. 38.
    Lee JS, Shin J, Park HM, Kim YG, Kim BG, Oh JW, et al. Liver extracellular matrix providing dual functions of two-dimensional substrate coating and three-dimensional injectable hydrogel platform for liver tissue engineering. Biomacromolecules. 2013;15:206–18.CrossRefGoogle Scholar
  39. 39.
    Feng ZQ, Chu XH, Huang NP, Leach MK, Wang G, Wang TC, et al. Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-I collagen-coated poly(L-lactic acid). Biomaterials. 2010;31:3604–12.CrossRefGoogle Scholar
  40. 40.
    Chua KN, Lim WS, Zhang P, Lu H, Wen J, Ramakrishna S, et al. Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold. Biomaterials. 2005;26:2537–47.CrossRefGoogle Scholar
  41. 41.
    Kim Y, Larkin AL, Davis RM, Rajagopalan PA. Comparative study of genome-wide transcriptional profiles of primary hepatocytes in collagen sandwich and monolayer cultures. Tissue Eng Part C Methods. 2010;16:1449–60.CrossRefGoogle Scholar
  42. 42.
    You J, Shin DS, Patel D, Gao Y, Revzin A. Characterizing the effects of heparin gel stiffness on function of primary hepatocytes. Adv Healthcare Mater. 2014;3:126–32.CrossRefGoogle Scholar

Copyright information

© The Regenerative Engineering Society 2017

Authors and Affiliations

  • Kirthanashri Srinivasan Vasanthan
    • 1
    • 2
  • Anuradha Subramanian
    • 1
  • Uma Maheswari Krishnan
    • 1
  • Swaminathan Sethuraman
    • 1
  1. 1.Centre for Nanotechnology & Advanced Biomaterials, School of Chemical & BiotechnologySASTRA UniversityThanjavurIndia
  2. 2.Department of BiotechnologySRM University HaryanaRajiv Gandhi Educational CityIndia

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